The present invention relates to new biologically active compounds which inhibit the cellular formation of niacinamide mononucleotide, which is one of the essential intermediates in the NAD(P) biosynthesis in the cell. The invention further concerns pharmaceutical compositions containing these compounds and their use, especially in the treatment of cancers, leukaemias or for immunosuppression. The invention also provides screening methods as a tool for detecting the above active compounds, and for examination of cell types with respect to their NAD(P) synthesis pathway.
NAD is synthesized in mammalian cells by three different pathways starting either from tryptophan via quinoline acid, from niacin (also referred to as nicotinic acid) or from niacinamide (also referred to as nicotinamide), as shown in FIG. 1.
The addition of a phosphoribosyl moiety results in the formation of the corresponding mononucleotides, niacin mononucleotide (dNAM) and niacinamide mononucleotide (NAM). Quinoline acid is utilized in a reaction with phosphoribosyl pyrophosphate (PRPP) to form niacin mononucleotide (dNAM). The enzyme catalyzing this reaction, quinoline acid phosphoribosyl transferase (), is found in liver, kidney and brain.
Niacin reacts with PRPP to form niacin mononucleotide (dNAM) The enzyme catalyzing this reaction is niacin phosphoribosyl transferase () and is widely distributed in various tissues. Both pathways starting either from tryptophan or from niacin as NAD precursors merge at the stage of the niacin mononucleotide formalin.
Niacinamide reacts with PRPP to give niacinamide mononucleotide (NAM). The enzyme that catalyses this reaction is niacinamide phosphoribosyl transferase (z,902 ). This enzyme is specific for niacinamide and is entirely distinct from niacin phosphoribosyl transferase (). It is also widely distributed in various tissues.
The subsequent addition of adenosine monophosphate to the mononucleotides results in the formation of the corresponding dinucleotides: Niacin mononucleotide and niacinamide mononucleotide react with ATP to yield niacin adenine dinucleotide (dNAD) and niacinamide adenine dinucleotide (NAD), respectively. Both reactions, albeit taking place on two different pathways, are catalyzed by the same enzyme, NAD pyrophosphorylase ().
A further amidation step is needed to convert niacin adenine dinucleotide (dNAD) to niacinamide adenine dinucleotide (NAD) The enzyme which catalyses this reaction is NAD synthetase (). NAD is the immediate precursor of niacinamide adenine dinucleotide phosphate (NADP). The reaction is catalyzed by NAD kinase (). For details see, for example, Cory, J. G. Purine and pyrimidine nucleotide metabolism. In: Textbook of Biochemistry and Clinical Correlations, 3rd edition, ed. Devlin, T., Wiley Brisbane 1992, pp 529-574.
Normal cells can typically utilize both precursors niacin and niacinamide for NAD(P) synthesis, and in many cases additionally tryptophan or its metabolites, which has been demonstrated for various normal tissues: Accordingly, Murine glial cells (cortex and hippocampus=brain) use: niacin, niacinamide, and quinoline acid (Grant et al. (1998), J. Neurochem. 70: 1759-1763). Human lymphocytes use niacin and niacinamide (Carson et al. (1987), J. Immunol. 138: 1904-1907; Berger et al. (1982), Exp. Cell Res. 137: 79-88). Rat liver cells use niacin, niacinamide and tryptophan (Yamada et al. (1983), Internat. J. Vit. Nutr. Res. 53: 184-191; Shin et al. (1995), Internat. J. Vit. Nutr. Res. 65: 143-146; Dietrich (1971) , Methods Enzymol. 18B: 144-149). Human erythrocytes use niacin and niacinamide (Rocchigiani et al. (1991), Purine and pyrimidine metabolism in man VII, Part B, ed. Harkness et al., Plenum Press, New York, pp 337-340. Leukocytes of guinea pigs use niacin (Flechner et al. (1970), Life Science. 9: 153-162).
NAD(P) is involved in a variety of biochemical reactions which are vital to the cell and have therefore been thoroughly investigated. This key function of NAD(P) has evoked also some investigations in the past on the role of this compound for the development and growth of tumors, and as to what the NAD(P) metabolism could also be utilized to combat tumors. Indeed, compounds aiming at the treatment of tumor diseases have been described which involvexe2x80x94concomitantly to other effectsxe2x80x94also the decrease of NAD(P) levels in the cell. However, these compounds primarily act by initiating the cellular synthesis of dinucleotide derivatives which structurally deviate from natural NAD. The biochemical consequences of this approach and the putative mechanisms of the resulting cell-damage are, therefore, manifold as outlined in the Table 1.
It is therefore not possible to make any predictions from these data on the biological effects of a primary and specific inhibition of the NAD biosynthesis in various cell types. In particular, it remains completely speculative whether this mechanism may be advantageous over the above utilization or dinucleotide derivatives with regard to tumor selectivity of the cell damaging effect, the most important feature of a potential drug for tumor therapy.
JP-459555, published in 1970, describes the extraction of a structurally unknown constituent from potatoes, baker""s yeast and bovine blood which inhibits respiration of tumor cells and NAD synthesis of erythrocytes. The inventors propose the use of this constituent for tumor therapy. However, the data presented in JP-459555 are far from making it clear or even probable that inhibition of NAD synthesis is useful for the therapy of cancer. The inventors rather come to the conclusion that the biological activity of the compound is multifaceted and not limited merely to the phenomenon of inhibition of NAD biosynthesis. In a study published later by the same research group (A. Kizu: Kyoto Furitsu Ika Daigaku Zasshi 80, pp. 14-24, 1971) it was shown that the extracted compounds (derivatives of glucose) inhibit respiration and glycolysis in tumor cells already within a few minutes in addition to inhibition of NAD synthesis. In fact, tumor cells treated with the extract for only 20 min suffered from such heavy damage that they did not grow in the abdominal cavity of mice in contrast to untreated control cells. In contrast to this finding, the present inventors have observed that compounds which promptly and selectively inhibit NAD synthesis in the cell exert a deleterious effect on tumor cells only after an exposure for 3-4 days, whereas an exposure for 20 min is completely ineffective irrespective of the concentration employed. Thus, it is very unlikely that it is NAD biosynthesis inhibition by which the extract disclosed in JP-459555 damages tumor cells. It is rather assumed that other mechanisms are primarily responsible for the cell death, while the reduction of the NAD levels is a secondary effect due to the general damage to the cell. The prompt deleterious effect on tumor cells as produced by this extract is, therefore, obviously due to a inhibition of cell respiration.
Also, the tumor preference of the cell killing effect of the extract, as described in JP-459555, can easily be explained by a characteristic feature of the respiration inhibiting effect of the extract, as this effect is marked in tumor cells but absent in liver cells. (FIG. 2 in A. Kizu). Thus, clearly JP-459555 did not disclose any means to affect tumor cells by NAD synthesis inhibition.
It was also known that DNA damaging cytotoxic compounds initiate a decrease of the cellular concentration of NAD. Some authors assumed that lowering of cellular NAD levels, with a resulting shortage of ATP within the cell, may play a role in the mechanism of cell death produced by these compounds (Daniel S. Martin and Gary K. Schwartz, Oncology Research, Vol. 9, pp. 1-5, 1997). The effect of these compounds on the NAD concentration within the cell results, however, indirectly from an enhanced NAD consumption by enzymes involved in DNA repair (see Table 1).
The primary effect of these compounds, namely damage to the DNA, has many consequences in addition to lowering cellular NAD levels. As known, the DNA is in control of the synthesis of many cellular constituents, like proteins and enzymes, which are of vital importance to the cell. Thus, the consequences of DNA damage are also manifold, lowering of the cellular NAD concentration being only one of them. The efficacy profile of a specific inhibition of the NAD biosynthesis can, therefore, not be concluded from observations made with these compounds.
Just as little information on what can be expected from a specific inhibition of the biosynthesis of NAD gives the symptomatology of niacinamide and niacin deficiency. These vitamins of the B group are precursors of the NAD biosynthesis as outlined above. Long term deficiency of these precursors results in a disease known as pellagra. Main symptoms are alterations of the skin and dementia. This syndrome shows no similarity to the chronic intoxication with any of the compounds discussed above.
WO 97/48695 describes new pyridyl alkane acid amines, methods for their production, medicaments containing these compounds as well as their use, especially in the treatment of tumor conditions and/or as cytostatic agents or as immunosuppressive agents. WO 97/48696 discloses new pyridyl alkene and pyridyl alkine acid amines, methods for their production, medicaments containing these compounds as well as their use, especially in the treatment of tumor conditions and/or as cytostatic agents or as immunosuppressive agents. In WO 97/48397 the use of pyridyl alkane, pyridyl alkene and/or pyridyl alkine acid amines, especially in the treatment of tumor conditions and/or as cytostatic agents or as immunosuppressive agents as well as medicaments with an amount of these compounds in combination with other cytostatic agents or immunosuppressive agents is disclosed. WO 99/31063, published Jun. 24, 1999, describes new piperazinyl-substituted pyridyl-alkane, alkene and alkine carboxamides with a saturated, one or several-fold unsaturated hydrocarbon residue in the carboxylic acid portion, methods for the synthesis of these compounds, medicaments containing these and their production as well as their therapeutic use especially as cytostatic agents and immunosuppresive agents, for example, in the treatment or prevention of various types of tumors and control of immune reactions, for example of autoimmune diseases. WO 99/31060, published Jun. 24, 1999, reports on new piperidinyl-substituted pyridyl-alkane, alkene and alkine carboxamides with a saturated or one or several-fold unsaturated hydrocarbon residue in the carboxylic acid portion, methods for the synthesis of these compounds, medicaments containing these and their production as well as their therapeutic use especially as cytostatic agents and immunosuppresive agents, for example, in the treatment or prevention of various types of tumors and control of immune reactions, for example of autoimmune diseases. New pyridylalkane, alkene and alkine carboxamides substituted with a cyclic imide and with a saturated or one or several-fold unsaturated hydrocarbon residue in the carboxylic acid group, methods for the synthesis of these compounds, medicaments containing these and their production as well as their therapeutic use especially as cytostatic agents and immunosuppresive agents, for example, in the treatment or prevention of various types of tumors inhibition of abnormal cell growth and control of immune reactions, for example of autoimmune diseases is the subject of WO 99/31087, published on Jun. 24, 1999. In WO 99/31064, published on Jun. 24, 1999, new pyridylalkane, alkene and alkine carboxamides substituted with a saturated, one or several-fold unsaturated hydrocarbon residue in the carboxylic acid grouping, methods for the synthesis of these compounds, medicaments containing these and their production as well as their therapeutic use especially as cytostatic agents and immunosuppresive agents, for example, in the treatment or prevention of various types of tumors and control of immune reactions, for example of autoimmune diseases are disclosed. All these applications disclose compounds or the use of compounds which have cytostatic activity and/or are useful in the treatment of tumor conditions, however neither do these applications give any indication that the biosynthesis of NAD is inhibited by these compounds nor do they implicitly disclose these compounds as being specific niacinamide phosphoribosyltransferase (NAPRT) inhibitors.
Thus, in summary, the state of the art does not allow to draw conclusions as to what can be expected from a primary and specific inhibition of the cellular synthesis of NAD because the compounds known to lower the cellular NAD concentration exert other primary effects which may affect cell survival by themselves. There exists no other reliable means to solve this question than the use of a specific inhibitor of NAD synthesis. But no such compound was available in the past.
Morton (R. K. Morton: Nature 181, pp. 540-543, 1958) proposes for human cancer therapy to aim at compounds which inhibit the NAD pyrophosphorylase (Enzyme  in FIG. 1) as the activity of this enzyme was assumed to be the limiting factor of NAD synthesis. Note that the biosynthesis pathway from both niacin and niacinamide, and also from tryptophan would be blocked by an inhibition of the NAD pyrophosphorylase since it acts on a late step of the biosynthesis pathway where the initially separated pathways starting from the different precursors tryptophan, niacin or niacinamide have already been united or are equally affected. No specific inhibitor of this enzyme has been found until now. Thus, no evidence for the correctness of this assumption is available.
The present invention is based on the surprising finding that in specific cell types utilization of niacinamide for cellular NAD(P) biosynthesis is of vital importance. Niacin or tryptophan which constitute alternative precursors in many other cell types investigated so far cannot be utilized, or at least not to an extent sufficient for cell survival. Accordingly, the present invention provides for biologically active compounds which inhibit the cellular formation of niacinamide mononucleotide. Compounds having this activity can easily be identified by the screening assay described below (also referred to hereinafter as NAPRT assay). Preferably, the present compounds at concentrations of xe2x89xa610 xcexcM exhibit an inhibitory activity on cellular NAD biosynthesis from the precursor niacinamide of at least 50%, more preferably at least 80% and most preferably at least 90% in such an assay.